Multi photon microscopy is a well-established technique in order to investigate biological tissue. It has the advantage over confocal fluorescence microscopy that it has a larger penetration depth in turbid media such as tissue, a smaller excitation load and a natural depth sectioning.
The disadvantage of non-linear imaging is the small absorption cross section of fluorophores for two photon excitation. This could be compensated by increasing the excitation power, but the increase of power is limited in practice to a damage threshold for so-called bleaching at around 10 mW average power in a submicron focus with a conventional laser system having a repetition rate of 80 MHz. The pulse energy should therefore be limited to below 1 nJ. Available laser systems, however, exhibit much larger average powers and consequently much larger pulse energies. The surplus of energy can be employed by splitting each pulse in a set of equidistant pulses with the same energy, preferably without loss of energy. By accelerating the repetition rate N-fold, one can generally:
increase the signal strength N-fold,
increase the data acquisition speed N-fold, or
reduce the photodamage probability, or
any combination thereof.
Various solutions exist for pulse splitters such as the use of Fabry-Perot etalons with two parallel, partially reflective mirrors, or the so-called optical rattler where a stack of parallel etalons is applied.
A recent monolithic pulse splitter has been disclosed by Na Ji, Jeffrey C. Magee & Eric Betzig (Nature Methods 5(2) 2008, pp 197-202 entitled “High-speed, low-photodamage nonlinear imaging using passive pulse splitters”). See also the corresponding US patent application US 2009/0067458, where an apparatus includes a pulsed laser source that produces a pulsed laser beam at an input repetition rate and an input pulse power, a passive pulse splitter (with two different materials interfaced with a 50% beam splitter) that receives the pulsed laser beam and outputs a signal including a plurality of sub-pulses for each input pulse of the pulsed laser beam, a sample, and a detector. The output signal has a repetition rate that is greater than the input repetition rate and the powers of each of the sub-pulses are less than the input pulse power.
The essence of the pulse splitter of Na Ji et al. is the fact that the two material layers have different refractive indices so that there is refraction at the interface with the 50% beam splitter. Materials with different refractive indices have the tendency to have different group velocity dispersions. This means that, when an ultra short pulse travels through the medium, not all optical frequencies that make up the pulse have the same velocity. Consequently, the pulse will broaden. This broadening effect is different for beams travelling through the two materials, i.e. medium 1 and medium 0. On the output ports of the pulse splitter, the pulses are mixed, resulting in alternatively more and less broadened pulses. Na Ji et al. proposes to use low dispersion materials, e.g. air and silica, to reduce the group velocity dispersion but this will only reduce the dispersion, not eliminate it, and this dispersion will also set a limit to the number of sub-pulses, N, and the available intra-pair pulse spacing time, Δt.
Hence, an improved pulse splitter device would be advantageous, and in particular a more efficient and/or reliable device would be advantageous.